Impact of environmental enrichment and allostatic load induced by repeated mixing on the transcriptome of three porcine brain tissues

Abstract Environmental enrichment and mixing represent two management procedures in pig production with opposing effects: while environmental enrichment can promote welfare by reducing stress, repeated mixing often induces stress and can negatively impact welfare. Despite their importance in pig husbandry, how these opposing management procedures affect brain function at the molecular level remains poorly understood. This study investigated how environmental enrichment and allostatic load induced by repeated mixing influence gene expression patterns across three porcine brain regions: hypothalamus, prefrontal cortex, and hippocampus. Using RNA sequencing, we analyzed brain tissues from 48 gilts subjected to a 2 × 2 factorial design comparing enrichment conditions (enriched vs. barren) and allostatic load (repeated vs. minimal mixing). While environmental enrichment showed minimal impact on gene expression, allostatic load resulted in 38 differentially expressed genes in the hippocampus, primarily involved in hormone regulation, protein transport, and neuronal activity. Weighted Gene Co-expression Network Analysis (WGCNA) identified modules moderately correlated with experimental conditions: six modules in hippocampus associated with allostatic load, and four modules in hypothalamus associated with enrichment. Functional annotation revealed that these modules were primarily enriched in biological processes related to synaptic signaling, neuron development, and metabolic activity. Hub genes from these modules were identified, and turned out not to overlap with differentially expressed genes. Our findings suggest that based on this experimental design, the hippocampus may be particularly responsive to stress-induced transcriptomic changes, while the molecular effects of the environmental enrichment appear more subtle. Overall, this study reveals limited but region-specific transcriptional responses to allostatic load in the porcine brain.